Electrofocusing of chicken liver dihydrofolic reductase purified by affinity chromatography on a Methotrexate-Sepharose column reveals multiple peaks of enzyme activity. The major peak is the "pure" enzyme devoid of bound substrate. At least two minor peaks may contain bound dihydrofolate and TPNH. The properties of the homogenous-substrate free enzyme have been examined by amino acid analysis, sulfhydryl group titration, methotrexate titration, ultracentrifugation, circular dichroism and fluorescence spectroscopy.